Three prototypical forms of retroviral reverse transcriptase have been studied thoroughly. Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase contains a single subunit of 78 kDa with RNA-dependent DNA polymerase and RNase H activity. This enzyme has been cloned and expressed in a fully active form in E. coli (reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 135 (1993)). Human Immunodeficiency Virus (HIV) reverse transcriptase is a heterodimer of p66 and p51 subunits in which the smaller subunit is derived from the larger by proteolytic cleavage. The p66 subunit has both an RNA-dependent DNA polymerase and an RNase H domain, while the p51 subunit has only a DNA polymerase domain. Active HIV p66/p51 reverse transcriptase has been cloned and expressed successfully in a number of expression hosts, including E. coli (reviewed in Le Grice, S. F. J., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory press, p. 163 (1993)). Within the HIV p66/p51 heterodimer, the 51-kD subunit is catalytically inactive, and the 66-kD subunit has both DNA polymerase and RNase H activity (Le Grice, S. F. J., et al., EMBO Journal 10:3905 (1991); Hostomsky, Z., et al., J. Virol. 66:3179 (1992)). Avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes but is not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase, is also a heterodimer of two subunits, alpha (approximately 62 kDa) and beta (approximately 94 kDa), in which alpha is derived from beta by proteolytic cleavage (reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1993), p. 135). ASLV reverse transcriptase can exist in two additional catalytically active structural forms, beta beta and alpha (Hizi, A. and Joklik, W. K., J. Biol. Chem. 252: 2281 (1977)). Sedimentation analysis suggests alpha beta and beta beta are dimers and that the alpha form exists in an equilibrium between monomeric and dimeric forms (Grandgenett, D. P., et al., Proc. Nat. Acad. Sci. USA 70:230 (1973); Hizi, A. and Joklik, W. K., J. Biol. Chem. 252:2281 (1977); and Soltis, D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA 85:3372 (1988)). The ASLV alpha beta. and beta beta reverse transcriptases are the only known examples of retroviral reverse transcriptase that include three different activities in the same protein complex: DNA polymerase, RNase H, and DNA endonuclease (integrase) activities (reviewed in Skalka, A. M., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1993), p. 193). The alpha form lacks the integrase domain and activity.
The conversion of mRNA into cDNA by reverse transcriptase-mediated reverse transcription is an essential step in many gene expression studies. However, the use of unmodified reverse transcriptase (RT) to catalyze reverse transcription is inefficient for a number of reasons. First, reverse transcriptase sometimes degrades an RNA template before the first strand reaction is initiated or completed, primarily due to the intrinsic RNase H activity present in reverse transcriptase. In addition, mis-priming of the mRNA template molecule can lead to the introduction of errors in the cDNA first strand. RTs have in fact been shown to incorporate one base error per 3000-6000 nucleotides for HIV RT, and 1/10,000 nucleotide for AMV RT during cDNA synthesis (Berger, S. L., et al., Biochemistry 22:2365-2372 (1983); Krug, M. S., and Berger, S. L., Meth. Enzymol. 152:316 (1987); Berger et al. Meth. Enzymol. 275: 523 (1996)). Secondary structure of the mRNA molecule itself may make some mRNAs refractory to first strand synthesis. Another factor which influences the efficiency of reverse transcription is the ability of RNA to form secondary structures. Such secondary structures can form, for example, when regions of RNA molecules have sufficient complementarity to hybridize and form double stranded RNA. Generally, the formation of RNA secondary structures can be reduced by raising the temperature of solutions which contain the RNA molecules. Thus, in many instances, it is desirable to reverse transcribe RNA at temperatures above 37° C. However, art known reverse transcriptases generally lose activity when incubated at temperatures much above 37° C. (e.g., 50° C.).
A variety of methods of attempting to engineer a thermostable reverse transcriptase are known in the art. These methods include using thermostable DNA polymerases that contain reverse transcriptase activity (Shandilya et al., Extremophiles, 2004 8:243), mutagenizing thermostable DNA polymerases to increase their reverse transcriptase activity (U.S. 2002/0012970), mutagenizing thermolabile reverse transcriptases (US 2004/0209276), using Mn2+ instead of Mg2+ in the presence of Taq/Tth DNA polymerases (Myers et al., Biochemistry 1991 30:7661), and using additives such as trehalose with thermolabile reverse transcriptases (Carninci et al., 1999 Proc Natl Acad Sci USA 95:520).
Scientists in the field have also tried different enzyme compositions and methods for increasing the fidelity of polymerization on DNA or RNA templates. For example, Shevelev et al., Nature Rev. Mol. Cell Biol. 3:364 (2002) provides a review on 3′-5′ exonucleases. Perrino et al., PNAS, 86:3085 (1989) reports the use of epsilon subunit of E. coli DNA polymerase III to increase the fidelity of calf thymus DNA polymerase α. Bakhanashvili, Eur. J. Biochem. 268:2047 (2001) describes the proofreading activity of p53 protein and Huang et al., Oncogene, 17:261 (1998) describes the ability of p53 to enhance DNA replication fidelity. Bakhanashvili, Oncogene, 20:7635 (2001) also reports that p53 enhances the fidelity of DNA synthesis by HIV type I reverse transcriptase. Hawkins et al. describes the synthesis of full length cDNA from long mRNA transcripts (2002, Biotechniques, 34:768).
U.S. Patent Application 2003/0198944A1 and U.S. Pat. No. 6,518,019 provide an enzyme mixture containing two or more reverse transcriptases (e.g., each reverse transcriptase having a different transcription pause site) and optionally one or more DNA polymerases. U.S. Patent Application 2002/0119465A1 discloses a composition that includes a mutant thermostable DNA polymerase and a mutant reverse transcriptase (e.g., a mutant Taq DNA polymerase and a mutant MMLV-RT). U.S. Pat. No. 6,485,917B1 and U.S. Patent application 2003/0077762 and EP patent application EP1132470 provide a method for synthesizing cDNA in the presence of an enzyme having a reverse transcriptional activity and an α-type DNA polymerase having a 3′-5′ exonuclease activity.
Removal of the RNase H activity of reverse transcriptase can eliminate the problem of RNA degradation of the RNA template and improve the efficiency of reverse transcription (Gerard, G. F., et al., FOCUS 11(4):60 (1989); Gerard, G. F., et al., FOCUS 14(3):91 (1992)). However such reverse transcriptases (“RNase H-” forms) do not address the additional problems of mis-priming and mRNA secondary structure.
There is a need in the art for a reverse transcriptase that exhibits increased stability.